The present invention concerns devices for the microscopic, histologic and/or molecular analysis of tissue specimens.
Biological mechanisms of many diseases have been clarified by microscopic examination of tissue specimens. Histopathological examination has also permitted the development of effective medical treatments for a variety of illnesses. In standard anatomical pathology, a diagnosis is made on the basis of cell morphology and staining characteristics. Tumor specimens, for example, can be examined to characterize the tumor type and predict whether the patient will respond to a particular form of chemotherapy. Although this microscopic examination and classification of tumors has improved medical treatment, the microscopic appearance of a tissue specimen stained by standard methods (such as hematoxylin and eosin) can often only reveal a limited amount of diagnostic or molecular information.
Recent advances in molecular medicine have provided an even greater opportunity to understand the cellular mechanisms of disease, and select appropriate treatments with the greatest likelihood of success. Some hormone dependent breast tumor cells, for example, have an increased expression of estrogen receptors on their cell surfaces, which indicates that the patient from whom the tumor was taken will likely respond to certain anti-estrogenic drug treatments. Other diagnostic and prognostic cellular changes include the presence of tumor specific cell surface antigens (as in melanoma), the production of embryonic proteins (such as xcex1-fetoprotein in liver cancer and carcinoembryonic glycoprotein antigen produced by gastrointestinal tumors), and genetic abnormalities (such as activated oncogenes in tumors). A variety of techniques have evolved to detect the presence of these cellular abnormalities, including immunophenotyping with monoclonal antibodies, in situ hybridization with probes, and DNA amplification using the polymerase chain reaction (PCR).
The development of new molecular markers, however, has been impeded by the inability to group a large number of tissues within a small surface area Only a limited amount of hybridoma supernatant may be available, particularly during the early phase of monoclonal antibody generation, which limits the number of specimens that can be analyzed. Even if large quantities of the immunohistologic agent are available, however, the reagents are expensive and may vary in reactivity. These problems led Battifora et al. to propose in Lab. Invest. 5:244-248 (1986), and in U.S. Pat. No. 4,820,504, that multiple tissue specimens may be grouped together on a single slide to enable the specimens to be simultaneously screened by application of a single drop of hybridoma supernatant. The specimens were prepared by using a hand-held razor blade to cut deparaffinized and dehydrated tissue specimens into slices, which were then bundled together randomly, wrapped in a sausage casing, and re-embedded in paraffin. This technique required a high degree of manual dexterity, and incorporated samples into a composite block in a manner that made it difficult to find and identify particular specimens of interest.
A modification of this process was disclosed by Wan et al., J. Immunol. Meth. 103:121-129 (1987). and Furmanski et al. in U.S. Pat. No. 4,914,022, in which cores of paraffin embedded tissue were obtained from standard tissue blocks. The cores were softened and straightened by manually rolling them on a warm surface and then bundled inside a conventional drinking straw. This method was said to be suitable for simultaneous histologic testing of multiple tissue specimens, for example in the characterization of monoclonal antibodies. The technique of Miller and Groothuis, A.J.C.P. 96:228-232 (1991) similarly rolled tissue strips into xe2x80x9clogsxe2x80x9d from which transverse sections were taken to be embedded in paraffin. The straw and log techniques, however, were labor intensive, required a high degree of manual dexterity, and also randomly arranged the samples in a manner that complicated the identification of specimens of interest Battifora and Mehta, Lab. Invest. 63:722-724 (1990), and U.S. Pat. No. 5,002.377, attempted to overcome some of the problems of random placement by cutting specimens into a plurality of narrow strips, which were individually positioned in parallel rectangular grooves in a mold. The tissue strips were embedded in agar gel that was poured into the grooves to produce a plate-like member with a series of ridges. Several of the ridged plates were stacked together and embedded in paraffin to form a tissue block. A similar approach was proposed by Sundblad, A.J.C.P. 102:192-193 (1993), in which the tissue strips were placed in triangular wedges instead of rectangular grooves. Slicing the tissue, assembling it into rows, and embedding it in several steps to form the block was a time-consuming method that reduced the efficiency of examining a large number of tissue specimens.
All of these techniques have been inadequate for the efficient preparation of an array of tissue specimens that can be used for rapid parallel analysis of a variety of independent molecular markers. This inefficiency has been a significant problem in fields such as cancer research, because cancer development and progression is a multi-step process that involves sequential losses, rearrangements and amplifications of several chromosomal regions and multiple genes. These events lead to a dysregulation of critical signal transduction pathways for cell growth, death, and differentiation. The details of this complex process remain incompletely understood, partly because high-throughput strategies and techniques for analyzing such genetic changes in large numbers of uncultured human tumors have not been available.
For example, simultaneous analysis of several genes within the same or related signal transduction pathways may be necessary to pinpoint critical, rate-limiting steps in the dysregulation of cancer cell growth. Furthermore, analysis of structural and numerical changes affecting several chromosomes, loci and genes at the same time may be needed to understand the patterns of accumulation of genetic changes in different stages of the cancer progression. Finally after novel genes and genetic changes of potential importance in cancer have been identified, substantial additional research is usually required to determine the diagnostic, prognostic and therapeutic significance of these molecular markers in clinical oncology.
Since the amount of tissue often becomes rate limiting for such studies, the ability to efficiently procure, fix, store and distribute tissue for molecular analysis in a manner that minimizes consumption of often unique, precious tumor specimens is important. It is therefore an object of this invention to perform large-scale molecular profiling of tissue specimens (such as tumors) with minimal tissue requirements, in a manner that allows rapid parallel analysis of molecular characteristics (such as gene dosage and expression) from hundreds of morphologically controlled tumor specimens.
The foregoing objects are achieved by a method of parallel analysis of tissue specimens, in which a plurality of donor specimens are placed in assigned locations in a recipient array, and a plurality of sections are obtained from the recipient array so that each section contains a plurality of donor specimens that maintain their assigned locations. A different histological analysis is performed on each section, to determine if there are correlations between the results of the different analyses at corresponding locations of the array. In particular embodiments, the donor specimen is obtained by boring an elongated sample, such as a cylindrical core, from donor tissue, and placing the donor specimen in a receptacle of complementary shape, such as a cylindrical core, in the recipient array. Analyses that may be performed on the donor specimens include immunological analysis, nucleic acid hybridization, and clinicopathological characterization of the specimen.
In a more particular embodiment of the method, a recipient block is formed from a rigid embedding medium such as paraffin that can be cut with a punch or microtome, and a separate donor block is also formed by embedding a biological specimen in the embedding medium. Cylindrical receptacle cores are bored in the recipient block to form an array of receptacles at fixed positions, and cylindrical donor sample cores are obtained from the embedded biological specimen in the donor blocks The donor sample cores are then placed in the cylindrical receptacles at assigned locations in the array, and the recipient block is sliced to obtain a cross-section of the donor sample cores in the array, without disrupting the assigned array locations. A different histological analysis may be performed on each section, for example by using different monoclonal antibodies that recognize distinct antigens, or a combination of antigenically distinct monoclonal antibodies and nucleic acid (e.g. RNA and DNA) probes on sequential sections. The result of each distinct histological analysis in each position of the array is compared, for example to determine if a tissue that expresses an estrogen receptor also has evidence that a particular oncogene has been activated.
In a more particular embodiment of the method, a recipient block is formed from a rigid embedding medium such as paraffin that can be cut with a punch or microtome, and a separate donor block is also formed by embedding a biological specimen in the embedding medium. Cylindrical receptacle cores are bored in the recipient block to form an array of receptacles at fixed positions, and cylindrical donor sample cores are obtained from the embedded biological specimen in the donor block. The donor sample cores are then placed in the cylindrical receptacles at assigned locations in the array, and the recipient block is sliced to obtain a cross-section of the donor sample cores in the array, without disrupting the assigned array locations. A different histological analysis may be performed on each section, for example by using different monoclonal antibodies that recognize distinct antigens, or a combination of antigenically distinct monoclonal antibodies and nucleic acid (e.g. RNA and DNA) probes on sequential sections. The result of each distinct histological analysis in each position of the array is compared, for example to determine if a tissue that expresses an estrogen receptor also has evidence that a particular oncogene has been activated. The presence or absence of the estrogen receptor and oncogene can then be correlated with clinical or pathological information about the tissue (such as the presence of metastatic disease or the histological grade of a tumor). This simultaneous parallel analysis of multiple specimens helps clarify the inter-relationship of multiple molecular and clinical characteristics of the tissue.
The invention also includes a method of obtaining small elongated samples of tissue from a tissue specimen, such as a tumor, and subjecting the specimen to laboratory analysis, such as histological or molecular analysis. The elongated tissue sample can be taken from a region of interest of the tissue specimen, and the size of the sample is small enough that the characterstic being analyzed is substantially homogenous throughout the small sample. In a disclosed embodiment, the sample is a cylindrical sample punched from the tissue specimen, wherein the cylindrical specimen is about 14 mm long, and has a diameter of about 0.14 mm, for example about 0.3-2.0 mm. In specific embodiments, the cylinder diameter is less than about 1.0 mm, for example 0.6 mm. The sample is preferably preserved in a manner (such as ethanol fixation) that does not interfere with analysis of nucleic acids, and the sample can therefore be subjected to any type of molecular analysis, such as any type of molecular analysis based on isolated DNA or RNA.
The invention also includes an apparatus for preparing specimens for parallel analysis of sections of biological material arrays. The apparatus includes a platform, a tissue donor block on the platform, and a punch that punches or bores a tissue specimen from the donor block. The platform can also carry a recipient block in which the punch forms an array of receptacles at selected positions. Each receptacle can be positioned so that a tissue specimen can be expelled from the reciprocal punch into the receptacle. An x-y positioning device incrementally moves the punch or recipient block with respect to one another as the punch reciprocates, to form the receptacle array. The x-y positioning device also aligns sequential receptacles of the recipient block with the punch to deliver tissue specimens from the punch into the receptacle. A stylet may be introduced into the punch to expel the contents of the punch, which may be either paraffin from the recipient block or tissue from the donor block. Regions of interest of the tissue specimen are located by positioning a thin section slide over the donor block, to align structures of interest in the thin section slide with corresponding tissue specimen regions in the donor block.
The invention also includes a computer implemented system for parallel analysis of consecutive sections of tissue arrays, in which an x-y positioning platform moves a tray to a plurality of coordinates that correspond to positions in a recipient block array. A receptacle punch then punches a receptacle core from a recipient block on the positioning platform, and a stylet expels the receptacle core from the receptacle punch. A donor punch (which may be the same or separate from the recipient punch) punches a donor specimen from a donor block on the positioning platform, and a stylet expels the donor specimen from the donor punch into the receptacle as the donor punch is introduced into the receptacle. The donor specimen suitably has a diameter that is substantially the same as the diameter of the receptacle, so that the donor specimen fits securely in the receptacle. The computer system identifies the tissue by its location in the recipient array, so that when the donor block is sectioned a corresponding position in each sectional array will contain tissue from the identical donor specimen.